Project Summary/Abstract Ubiquitin-dependent proteolysis plays a significant role in various physiological processes including cell cycle control and cellular proliferation and is frequently the target of oncogenic transfomation. The specficity of protein substrate ubiquitylation is dictated by the activity of one of many E3 ubiquitin ligases either directly or through substrate recognition through adaptor proteins. Among these, the Cullin 4-based E3 ubiquitin ligase (CRL4) is emerging as a master regulator of cellular proliferation and genomic stability and is involved in multiple DNA repair processes. Cdt2/DTL, a WD-repeat containing protein associates with CRL4 (CRL4-Cdt2) and functions as a substrate recognition factor for recuiting substrates to the rest of the CRL4 ubiquitin ligase. CRL4-Cdt2 has recently been shown to promote the ubiquitin-dependent destruction of the replication initiation protein Cdt1 and the cyclin-dependent kinase (CDK) inhibitor p21, both in S-phase of the cell cycle and in response to UV irradiation. Significantly, Cdt2 is frequently overexpressed in a variety of human tumors and its expression correlates with tumor grade, metastasis and poor survival. This study aims at understanding how CRL4-Cdt2 impacts on genomic stability and contributes to cancer development. Specifically, I will A) Identify and characterize novel substrates for the CRL4-Cdt2 E3 ubiquitin ligase complex. B) Identify the mechanism by which Cdt2 negatively regulates p21 transcription and C) Test whether Cdt2 exhibits oncogenic activity in vivo. Using tap-tandem purification and mass-spectrometry analysis of Cdt2-associated proteins from human cells with or without DNA damage, I will identify new CRL4-Cdt2 substrates that may be involved in cellular proliferation and/or DNA repair. Standard biochemical techniques, including in vivo and in vitro ubiquitylation assays using purfied CRL4-Cdt2 E3 ubiquitin ligase complexes, will verify whether the identified proteins are bona fide substrates. I will also generate transgenic mice overexpressing Cdt2 from Cre/LoxP constructs and test whether Cdt2 overexpression contributes to tumor development in animal model system. The results will advance our understanding of how to exploit the regulated process of protein ubiquitylation and proteolysis for cancer intervention purposes.